🧬 PubMed RNA Editing Daily Digest

Effective antiviral drugs remain unavailable for many clinically relevant pathogens, including the hepatitis E virus (HEV). This study aimed to evaluate the CRISPR/Cas13d system as a potential antiviral strategy against HEV. We developed a reporter assay to screen CRISPR RNAs (crRNAs) targeting conserved regions of the HEV genome and tested their antiviral activity in human hepatoma cells using a robust HEV cell culture model. HEV replication was assessed using a subgenomic replicon, infectious particle production was quantified by immunofluorescence and titration assays. A bioinformatic analysis was performed to identify a minimal set of crRNAs capable of broadly targeting circulating human pathogenic HEV strains. A crRNA screen identified multiple functional crRNAs targeting HEV-3, with ORF1-targeting crRNAs significantly reducing viral capsid expression (p < 0.01) and decreasing the number of HEV-infected cells (p < 0.01). Cas13d-mediated targeting led to robust reduction of HEV replication and markedly lowered infectious virus production in vitro (p < 0.001). Bioinformatic analysis revealed that just three distinct crRNAs could cover ∼95% of known HEV genomes with zero mismatches, while four crRNAs achieved complete coverage. Our findings demonstrate that CRISPR/Cas13d can target HEV replication and viral progeny production in vitro. The identification of a minimal crRNA set capable of broadly targeting circulating HEV strains suggests that the CRISPR/Cas13d system may offer an antiviral strategy to address challenges related to viral evolution and treatment escape. This study establishes CRISPR/Cas13d as a proof-of-concept antiviral strategy against hepatitis E virus (HEV), demonstrating suppression of viral replication and particle production in vitro. By identifying a minimal set of broadly effective crRNAs, we provide a framework for targeting diverse HEV variants and buffering against viral evolution. These findings highlight the potential of CRISPR-based systems as innovative antiviral strategies.

Simian immunodeficiency viruses (SIVs) have crossed from apes to humans at least four times, but only one event gave rise to the AIDS pandemic. The host barriers that pandemic HIV-1 group M ( Four independent transmission events of simian immunodeficiency viruses from chimpanzees and gorillas to humans gave rise to human immunodeficiency virus type 1, but only one led to the global AIDS pandemic. Understanding which adaptations allowed the pandemic HIV-1 M strains to spread efficiently in humans remains a key question in virus evolution and public health. In this study, we engineered replication-competent SIVcpz constructs carrying more than 1,500 single-guide RNAs to identify antiviral genes in Cas9-expressing cells. This approach revealed several cellular factors that restrict SIVcpz but not the pandemic HIV-1 M strains analyzed in primary human T cells. These findings provide new insights into antiviral defense mechanisms and the adaptations that most likely contributed to the efficient spread of HIV-1.

Incomplete editing and chimeric phenotypes are major challenges in CRISPR/Cas9-mediated genome editing of polyploid crops. In this study, a single guide RNA (gRNA) was designed to target a conserved dinucleotide-binding motif within exon 3 of the phytoene desaturase (PDS) gene in 'Grand Naine' banana. The gRNA was carefully selected for GC content, guanine residues near the PAM, and predicted secondary structure to enhance Cas9 cleavage efficiency. Agrobacterium-mediated transformation of embryonic cell suspensions produced 102 putative transgenic plants, all exhibiting altered phenotypes, with 91% displaying albino and 9% pale green coloration, indicating efficient PDS gene knockout and absence of chimerism. Sequencing confirmed tri-allelic editing, with all edited plants consistently showing two identical and one distinct mutation. Notably, small in-frame deletions of two to six amino acids within the conserved motif were sufficient to abolish PDS function, confirming its critical role in carotenoid biosynthesis. This strategy is adaptable to clonally propagated polyploid crops, providing a practical framework for achieving high-efficiency, uniform genome edits and supporting the development of precise, non-chimeric CRISPR/Cas9 editing approaches.